U.S. patent number 6,130,094 [Application Number 09/152,009] was granted by the patent office on 2000-10-10 for reagents including a carrier and fluorescent labeling complexes with large stokes shift formed by coupling together cyanine and other fluorochromes capable of resonance energy transfer.
This patent grant is currently assigned to Carnegie Mellon University. Invention is credited to Ratnakar B. Mujumdar, Swati R. Mujumdar, Alan S. Waggoner.
United States Patent |
6,130,094 |
Waggoner , et al. |
October 10, 2000 |
**Please see images for:
( Certificate of Correction ) ** |
Reagents including a carrier and fluorescent labeling complexes
with large stokes shift formed by coupling together cyanine and
other fluorochromes capable of resonance energy transfer
Abstract
The present invention provides low molecular weight fluorescent
labeling complexes with large wavelength shifts between absorption
of one dye in the complex and emission from another dye in the
complex. These complexes can be used, for example, for
multiparameter fluorescence cell analysis using a single excitation
wavelength. The low molecular weight of the complex permits
materials labeled with the complex to penetrate cell structures for
use as probes. The labeling complexes are synthesized by covalently
attaching through linkers at least one cyanine fluorochrome to
another low molecular weight fluorochrome to form energy
donor-acceptor complexes. Resonance energy transfer from an excited
donor to fluorescent acceptor provides wavelength shifts up to 300
nm. The fluorescent labeling complexes preferably contain reactive
groups for the labeling of functional groups on target compounds,
such as derivatized oxy and deoxy polynucleic acids, antibodies,
enzymes, proteins and other materials. The complexes may also
contain functional groups permitting covalent reaction with
materials containing reactive groups.
Inventors: |
Waggoner; Alan S. (Pittsburgh,
PA), Mujumdar; Swati R. (Glenshaw, PA), Mujumdar;
Ratnakar B. (Glenshaw, PA) |
Assignee: |
Carnegie Mellon University
(Pittsburgh, PA)
|
Family
ID: |
23893630 |
Appl.
No.: |
09/152,009 |
Filed: |
September 11, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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476880 |
Jun 7, 1995 |
6008373 |
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Current U.S.
Class: |
436/63;
252/183.11; 252/700; 435/6.12; 435/7.1; 436/800; 536/22.1;
536/23.1 |
Current CPC
Class: |
G01N
33/533 (20130101); G01N 33/582 (20130101); C09B
11/22 (20130101); C09B 23/06 (20130101); C09B
23/083 (20130101); C09B 23/086 (20130101); C09B
23/10 (20130101); Y10S 436/80 (20130101) |
Current International
Class: |
G01N
33/58 (20060101); G01N 33/533 (20060101); G01N
033/546 (); G01N 027/26 (); G01N 033/533 (); G01N
033/58 (); C12Q 001/68 () |
Field of
Search: |
;544/212,328
;546/272,273 ;548/427,455 ;435/6,7.1 ;436/800,63 ;536/22.1,23.1
;935/77 ;252/700,183.11 |
References Cited
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May 1988 |
Recktenwald et al. |
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Haugland et al. |
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Recktenwald et al. |
4900686 |
February 1990 |
Arnost et al. |
5248782 |
September 1993 |
Haugland et al. |
5268486 |
December 1993 |
Waggoner et al. |
5274113 |
December 1993 |
Kang et al. |
5332662 |
July 1994 |
Ullman |
5378634 |
January 1995 |
Nobuyuki et al. |
5453505 |
September 1995 |
Lee et al. |
5654419 |
August 1997 |
Mathies et al. |
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Other References
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Amos Carmel, et al., Use of Substrates with Fluorescent Donor and
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No. 2, pp. 105-111, 1993. .
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|
Primary Examiner: Higel; Floyd D.
Attorney, Agent or Firm: Kirkpatrick & Lockhart LLP
Parent Case Text
This is a divisional application of application Ser. No. 08/476,880
filed Jun. 7, 1995, now U.S. Pat. No. 6,008,373.
Claims
What we claim is:
1. A reagent comprising:
a fluorescent water soluble labeling complex consisting of:
(i) one or more low molecular weight first fluorochromes, each
having first absorption and emission spectra, covalently attached
through a linker to one or more low molecular weight second
fluorochromes, each having second absorption and emission spectra,
said linker being between 2 to 20 bond lengths and wherein at least
one of said first and second fluorochromes is a cyanine dye and
wherein the wavelength of the emission maximum of at least one said
second fluorochrome is longer than the wavelength of the emission
maximum of at least one said first fluorochrome and a portion of
the absorption spectrum of at least one said second fluorochrome
overlaps a portion of the emission spectrum of at least one said
first fluorochrome for transfer of energy absorbed by said first
fluorochrome upon excitation with light to said second
fluorochrome;
(ii) at least one bonding group capable of forming a covalent bond
with a carrier material; and,
(iii) at least one water solubilizing constituent attached to said
complex, said water solubilizing constituent being unreactive with
said at least one bonding group,
having covalently bonded thereto a carrier material having a group
that reacts with said bonding group of said complex.
2. The reagent recited in claim 1 wherein said carrier material has
a functional group selected from the group consisting of amino,
sulfhydryl, carbonyl, hydroxyl and carboxyl and said carrier
material is selected from the group consisting of antibody,
protein, nucleotide derivatized to contain one of an amino,
sulfhydryl, carbonyl, carboxyl, or hydroxyl groups, and oxy or
deoxy polynucleic acids derivatized to contain one of an amino,
sulfhydryl, carbonyl, carboxyl or hydroxyl groups.
3. The reagent recited in claim 1 wherein said first fluorochrome
has an extinction coefficient greater than 20,000 Liters per mole
centimeter.
4. The reagent recited in claim 1 wherein said second fluorochrome
has a fluorescence quantum yield greater than or equal to 0.05.
5. The reagent recited in claim 1 wherein said water solubilizing
constituents are selected from the group consisting of amide,
sulfonate, sulfate, phosphate, quaternary ammonium, hydroxyl and
phosphonate.
6. The reagent recited in claim 1 wherein one of said bonding group
or said carrier group is a reactive group selected from the group
consisting of succinimidyl ester, isothiocyanate, isocyanate,
iodoacetamide, acid halide, carbodiimide, substituted
hydroxylamines, substituted hydrazine, dichlorotriazine, maleimide,
sulfonyl halide, alkylimidoester, arylimidoester and
phosphonamidite and the other is a functional group reactive with
said reactive group.
7. The reagent recited in claim 1 wherein there is one second
fluorochrome and there are a plurality of said first fluorochromes
each covalently attached through a linker to said second
fluorochrome and each being capable, upon excitation with light, of
transferring energy to said second fluorochrome.
8. The reagent recited in claim 1 wherein there is one first
fluorochrome and there are a plurality of said second fluorochromes
each covalently attached through a linker to said first
fluorochrome and each being capable of accepting energy from said
first fluorochrome when said first fluorochrome is excited by
light.
9. The reagent recited in claim 1 further comprising:
a third fluorochrome having third absorption and emission spectra
covalently attached to said second fluorochrome;
the wavelength of the emission maximum of said third fluorochrome
being loner than the wavelength of the emission maximum of said
second fluorochrome, and a portion of the emission spectrum of said
second fluorochrome overlapping a portion of the absorption
spectrum of said third fluorochrome for transferring energy
absorbed from said first fluorochrome by said second fluorochrome
to said third fluorochrome.
10. The reagent recited in claim 1 wherein the combined molecular
weight of said first and second fluorochromes of said complex is
within the range of about 500 to 10,000 Daltons.
11. The reagent recited in claim 1 wherein said carrier material is
selected from the group consisting of a polymer particle, proteins,
cells, nucleotides and nucleic acids.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to fluorescent labeling complexes,
and more particularly to low molecular weight fluorescent complexes
with large Stokes shifts.
2. Description of the Invention Background
Fluorescence labeling is an important technology for detecting
biological molecules. For example, antibodies can be labeled with
fluorescent dyes, The binding of antibodies to their specific
target molecules can then be monitored on the basis of a
fluorescence signal, which may be detected with a spectrometer,
immunofluorescence instrument, flow cytometer, or fluorescence
microscope. In a similar way, DNA sequences can be detected with
fluorescence detection instruments after the DNA has been
"hybridized" with a complementary DNA sequence that has been
labeled with a fluorescent dye.
Very bright and water soluble fluorescent labeling reagents are
important for sensitive detection of labeled antibodies, DNA
probes, ligands, cytokines, drugs, lipids, metabolites and other
molecules and compounds of interest. Multiparameter analysis using
fluorescent labels with distinctly different emission wavelengths
further increase the important of this technology by providing a
powerful tool for correlating multiple antigenic or genetic
parameters in individual cells. In epifluorescence microscopy, a
continuous light source with different sets of excitation and
emission filters are used to excite and detect each fluorescent
species. This approach works especially well if the absorption and
emission wavelengths of each of the fluorophores are relatively
close together (e.g. Stokes shifts of 15-30 nm). Most of the highly
fluorescent, low molecular weight fluorochromes like the cyanines
and xanthenes have narrow absorption and emission peaks and small
Stokes shifts. Up to 5 separate fluorescent labels have been
analyzed on the same specimen by microscopy using spifluorescence
filter sets as described in DeBiasio, R., Bright, G. R., Ernet, L.
A., Waggoner, A. S., Taylor, D. L. "Five-parameter fluorescence
imaging: Wound healing of living Swiss 3T3 cells," Journal of Cell
Biology, vol. 105, pp. 1613-1622 (1987).
Flow cytometers and confocal microscopes are different from
microscopes equipped with separate epifluorescence filter sets, in
that they utilize lasers with defined wavelengths for fluorescence
excitation. While it is easy to find a single fluorophore that can
be efficiently excited at a particular laser wavelength, it is
difficult to find additional fluorescent labels with large enough
Stokes shifts to provide emission well separated from that of the
first fluorophore. The naturally occurring phycobiliproteins are a
class of multichromophore fluorescent photosystem proteins that
have large wavelength shifts. See, Oi, V. T., Glazer, A. N.,
Stryer, L. "Fluorescent phycobiliprotein conjugates for analyses of
cells and molecules," Journal of Cell Biology, vol. 93, pp. 981-986
(1982). These can be covalently coupled to antibodies and have
become widely used in flow cytometry for 2 color lymphocyte subset
analysis. R-phycoerythrin (R-PE), a photosystem protein containing
34 bilin fluorophores which can be excited at 488 nm with the
widely available argon ion laser, has been especially useful. It
fluoresces maximally at 575 nm. R-PE and fluorescein can both be
excited at 488 nm, but R-PE can readily be discriminated with
optical band-pass interference filter sets from the fluorescein
signal, which appears at 525 nm. Recently, 3-color
immunofluorescence by flow cytometry has become possible through
the development of tandem conjugate labeling reagents that contain
a reactive cyanine fluorescent dye which is excited at 488 nm and
fluoresces at 613 nm, and is sold commercially under the name
Cychrome. See, U.S. Pat. No. 4,876,190 and Waggoner, A. S., Ernst,
L. A., Chen, C. H. , Rechtenwald, D. J., "PE-CY 5; A new
fluorescent antibody label for 3-color flow cytometry with a single
laser," Ann. NY Acad. Sci., vol. 677, pp. 185-193 (1993). With
these tandem fluorophores, energy transfer from excited R-PE to the
Texas Red or the reactive pentamethine cyanine known as CY5 leads
to fluorescence at 620 nm or 670 nm, respectively.
The phycobiliprotein-based labels are very fluorescent and provide
excellent signals in 2 and 3-parameter experiments for detection of
cell surface antigens. However, these reagents have not been widely
utilized for measurement of cytoplasmic antigens or for detection
of chromosomal markers by fluorescence in situ hybridization
because their large size (MW=210,000 Daltons) limits penetration
into dense cell structures.
There is a need for a new class of low molecular weight fluorescent
labels that will provide multicolor fluorescence detection using
single wavelength excitation. There is a further need for several
such fluorescent labels each of which can be excited optimally at a
particular laser wavelength but that fluoresce at significantly
different wavelengths.
SUMMARY OF THE INVENTION
The present invention provides a low molecular weight fluorescent
labeling complex which includes a first, or donor, fluorochrome
having first absorption and emission spectra, and a second, or
acceptor, fluorochrome having second absorption and emission
spectra. At least one of the first or second fluorochromes is a
cyanine dye. The wavelength of the emission maximum of the second
fluorochrome is loner than the wavelength of the emission maximum
of the first fluorochrome, and a portion of the absorption spectrum
of the second fluorochrome overlaps a portion of the emission
spectrum of the first fluorochrome for transfer of energy absorbed
by the first fluorochrome upon excitation with light to the second
fluorochrome.
The complex also includes a linker for covalently attaching the
fluorochromes to permit resonance energy transfer between the first
and the second fluorochromes. The linker may be flexible and in a
preferred embodiment, separates the fluorochromes by a distance
that provides efficient energy transfer, preferably better than
75%. The linker may be about 2 to 20 bond lengths. A preferred
length for the linker is less than 70 Angstroms (7 nm), and more
preferably, less than 20 Angstroms (2 nm). In the case of flexible
linkers, particularly when the labeling complexes are in solution,
the relative orientations of the first and second fluorochromes
changes as the linker flexes.
The first fluorochrome preferably has an extinction coefficient
greater than 20,000 Liters/mole cm and preferably greater than
50,000 Liters/mole cm and the second fluorochrome has a
fluorescence quantum yield greater than or equal to about 0.05.
Quantum yield is generally related to a molecule's rigidity or
planarity and indicates the molecules propensity to fluoresce, i.e.
to give off energy as light, rather than as heat when energy is
provided to the molecule. The combined molecular weight of the
fluorochromes and the linker is in the range of 500 to 10,000
Daltons.
The complex includes a target bonding group capable of forming a
covalent bond with a target compound to enable the complex to label
the target, such as a carrier material or a biological compound.
The target bonding group may be a reactive group for reacting with
a functional group on the target compound or molecule.
Alternatively, the complex may contain the functional group and the
target may contain the reactive constituent. The reactive group is
preferably selected from the group consisting of succinimidyl
ester, isothiocyanates, dichlorotriazine, isocyanate,
iodoacetamide, maleimide, sulfonyl halide, acid halides,
alkylimidoester, arylimidoester, substituted hydrazines,
substituted hydroxylamines, carbodiimides, and phosphoramidite. The
functional group may be selected from the group consisting of
amino, sulfhydryl, carboxyl, hydroxyl and carbonyl. The target may
be antibody, antigen, protein, enzyme, nucleotide derivatized to
contain one of an amino, hydroxyl, sulfhydryl, carboxyl or carbonyl
groups, and oxy or deoxy polynucleic acids derivatized to contain
one of an amino, hydroxy, sulfhydryl, carboxyl or carbonyl groups,
cells, polymer particles or glass beads. In the alternative
embodiment, the target may be derivatized to contain the reactive
groups identified above to form covalent bonds with the functional
groups on the complex.
The complex preferably also includes water solubilizing
constituents attached thereto for conferring a polar characteristic
to the complex. They are preferably attached to the aromatic ring
of the cyanine fluorochrome. The water solubilizing constituents
must be unreactive with the target bonding group of the complex.
The solubilizing constituents are preferably selected from the
group consisting of amide, sulfonate, sulfate, phosphate,
quaternary ammonium, hydroxyl and phosphonate. Water solubility is
necessary when labeling protein and oxy or deoxy nucleic acids
derivatized with amino groups or sulfhydryl groups in aqueous
solutions. A less polar form of the energy transfer compound may
bind noncovalently to DNA by intercalation between base pairs or by
interaction in the minor groove of DNA. Such compounds would be
useful for DNA quantification or localization.
In addition to the embodiment of the invention which includes a
single donor and a single acceptor fluorochrome, the fluorescent
labeling complex may further include a third fluorochrome having
third absorption and emission spectrum and covalently attached to
the second fluorochrome. The wavelength of the emission maximum of
the third fluorochrome is longer than the wavelength of the
emission maximum of the second fluorochrome, and a portion of the
emission spectrum of the second fluorochrome overlaps a portion of
the absorption spectrum of the third fluorochrome for transferring
energy absorbed from the first fluorochrome to the second
fluorochrome to the third fluorochrome. Energy transfer procedes
consecutively, i.e. in series, from the first to the second to the
third fluorochromes.
In an alternative embodiment, the complex may include a plurality
of the first fluorochromes each covalently linked to the second
fluorochrome and each capable, upon excitation with light, of
transferring energy to the second fluorochrome. In another
embodiment, the complex may include a plurality of the second
fluorochromes each covalently linked to the first fluorochrome and
each capable of accepting energy from the first fluorochrome when
the first fluorochrome is excited by light. The plurality of first
or second fluorochromes may be the same molecule or may be
different. For example, there may be several donor fluorochromes
which are excitable at different wavelengths to accommodate
different excitation light sources. Energy transfer procedes in
parallel in these embodiments.
The labeling complexes of the invention are synthesized preferably
by covalently linking cyanine fluorochromes to other cyanine
fluorochromes to form energy donor-acceptor complexes. Cyanine
fluorochromes are particularly useful for preparation of these
complexes because of the wide range of spectral properties and
structural variations available. See, for example, Mujumdar, R. B.,
Ernst, L. A., Mujumdar, S. R., Lewis, C., Waggoner, A. S. "Cyanine
dye labeling reagents. Sulfoindocyanine succininmidyl ester,"
Bioconjugate Chemistry, vol. 4, pp. 105-111 (1993) and U.S. Pat.
No. 5,268,486 to Waggoner et al., the disclosure of which is
incorporated herein by reference.
The invention also includes a reagent and a method for making the
reagent including incubating the fluorescent water soluble labeling
complex described above with a carrier material. One of the complex
or the carrier material has a functional group that will react with
a reactive group of the other of the complex or the carrier
material to form a covalent bond
there between. The carrier material can be selected from the group
consisting of polymer particles, glass beads, cells, antibodies,
antigens, protein, enzymes, nucleotide derivatized to contain one
of an amino, sulfhydryl, carbonyl, carboxyl or hydroxyl groups, and
oxy or deoxy polynucleic acids derivatized to contain one of an
amino, sulfhydryl, carboxyl, carbonyl or hydroxyl groups.
Alternatively, the carrier material may contain the reactive groups
and the fluorescent labeling complex of the invention may contain
any of the aforementioned functional groups that will react with
the reactive groups to form covalent bonds.
In an alternative embodiment, the fluorescent complexes of the
invention need not have a reactive group when used to noncovalently
bind to another compound. For example, the complex may be
dissolved, then mixed in an organic solvent with a polymer
particle, such as polystyrene, then stirred by emulsion
polymerization. The solvent is evaporated and the fluorescent dye
complex is absorbed into the polystyrene particles.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be better understood by reference to the
drawings in which:
FIG. 1 is a schematic illustration of the overlapping absorption
and emission spectra of four cyanine fluorochromes that can be used
in the energy transfer labeling complex of the present
invention;
FIG. 2(a) is the absorbance spectrum for the complex consisting of
a trimethine and a pentamethine cyanine in a 1:1 ratio in methanol
and FIG. 2(b) is the absorbance spectrum for the complex consisting
of a trimethine and a pentamethine cyanine in a 2:1 ratio in
methanol;
FIG. 3 illustrates the absorption spectra of two fluorescent
labeling complexes, complex 1 (solid) in methanol, comprised of one
cyanine donor and one cyanine acceptor, and complex 6 (dotted) in
methanol, comprised of two cyanine donors and one cyanine
acceptor;
FIGS. 4(a) and (b) illustrate the absorbance (solid) and
fluorescence (dotted) spectra of complex 1 of the invention made of
trimethine and pentamethine cyanine dyes in (a) methanol and (b)
PBS;
FIG. 5 illustrates a normalized excited spectra of the complex 1 in
PBS (solid line), methanol ( ), glycerol ( ), and complex
1--streptavidin conjugate in PBS (----);
FIG. 6 illustrates the absorbance spectra in PBS of Sheep
IgG-complex 1 conjugates at various dye:protein ratios
demonstrating that no dimer formation involving either donor or
acceptor is evident with increasing dye:protein ratios; and,
FIG. 7 illustrates the two color flow cytometry analysis of human
lymphocytes labeled with anti-CD4-PE and
anti-CD3-streptavidin--complex 1 to mark the helper cell subset of
T-cells and total T-cell subset, respectively, showing a subset of
complex 1 labeled cells without the PE signal and a second subset
of complex 1 labeled cells that is PE stained;
FIG. 8 is the absorbance spectrum for the complex consisting of
fluorescein and a trimethine cyanine;
FIG. 9 is the fluorescence spectrum for the complex of FIG. 8;
FIG. 10 is the absorbance spectrum for the complex consisting of
fluorescein, a trimethine cyanine and a pentamethine cyanine;
FIG. 11 is the fluorescence spectrum for the complex of FIG.
10;
FIG. 12 is the absorbance spectrum for the complex consisting of
fluorescein, a trimethine cyanine and a heptamethine cyanine;
and,
FIG. 13 is the fluorescence spectrum for the complex of FIG.
12.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention provides low molecular weight, preferably
water soluble, fluorescent labeling complexes with large
excitation-emission wavelength shifts or Stokes shifts. For
purposes of the present specification, the Stokes shift of the
fluorescent complex is the absolute difference in nanometers
between the absorbance maximum of the lowest light absorber of the
complex and the fluorescence of the longest wavelength emitter of
the complex. The complexes, as briefly explained above, contain two
or more fluorochromes linked together for transfer of energy from a
shorter wavelength to a loner wavelength. As shown schematically in
FIG. 1, the first, donor fluorochrome absorbs energy upon
excitation at an excitation wavelength (solid line) within its
absorbance spectrum and emits energy at a wavelength within its
emission spectrum (broken line). When linked at an appropriate
orientation to a second, acceptor fluorochrome, the donor
fluorochrome transfers, or donates, the energy of its excited state
to the acceptor fluorochrome at a wavelength within the absorbance
spectrum (solid line) of the acceptor fluorochrome. The acceptor
fluorochrome absorbs the donated energy and emits it at a
wavelength within its emission spectrum (broken line), which as
shown, is loner than the longest wavelength of the emission spectra
of the donor fluorochrome. It is important, therefore, that the
emission spectrum of the donor fluorochrome overlap with the
absorption spectrum of the acceptor fluorochrome. The overlapping
areas are shown by hatched lines. The greater the overlap, the more
efficient the energy transfer.
The complexes include at least one cyanine fluorochrome, and
preferably at least one polymethine cyanine dye. The cyanines are
particularly useful due to the wide range of spectral properties
and structural variations available. Several such complexes will be
described for purposes of this detailed description. Other low
molecular weight fluorochromes in addition to the cyanine
fluorochromes, such as the fluoresceins, pyrene trisulfonates,
which are sold under the trade mark cascade blue, rhodamines and
derivatives of the bispyrromethene boron-difluoride dyes, such as
3,3',5,5'-tetramethyl 2,2'-pyromethene-1,1'-boron-difluoride, sold
under the trademark BODIPY by Molecular Probes, Inc., can be used
to form the fluorescent labeling complexes of the invention. BODIPY
analogs are disclosed in U.S. Pat. Nos. 4,774,339, 5,187,223,
5,248,782 and 5,274,113, all to Hougland and Kang, as well as in
"Handbook of Fluorescent Probes and Research Chemicals" compiled by
Hougland and published by Molecular Probes, Inc.
The fluorescent labeling complexes of the invention have low
molecular weights and can be readily conjugated to antibodies,
other proteins, and DNA probes. Low molecular weight as used herein
shall mean that the combined molecular weight of the fluorochromes
and linker of the complex is between about 500 and 10,000 Daltons,
and for a two fluorochrome complex, preferably in the range of 1000
to 2500 Daltons. Therefore, these labeled species will have much
greater penetration into intracellular environments than is
possible with the large phycobiliprotein labels currently in use.
The low molecular weight fluorescent labeling complexes of the
invention should be valuable not only for flow cytometry, but also
for laser confocal microscopy and for other detection systems
requiring multicolor detection with single wavelength
excitation.
Many structural varieties and modifications of cyanines are
possible. By varying the number of carbons in the methine bridge of
the cyanine dyes and the heteroatoms or other constituents on the
cyanine dyes, a variety of different spectral qualities can be
achieved. The cyanine dyes are especially well adapted to the
analysis of a mixture of components wherein dyes of a variety of
emission wavelengths are required because specific cyanine and
related dyes can be synthesized having a wide range of excitation
and emission wavelengths. Specific cyanine and related dyes have
specific excitation and emission wavelengths can be synthesized by
varying the number of methine groups or by modifying the cyanine
ring structures. In this manner, it is possible to synthesize dyes
having particular excitation wavelengths to correspond to a
particular excitation light source, such as a laser, e.g., a HeNe
laser or a diode laser. Therefore, energy transfer labels can be
made that absorb and emit efficiently at most wavelengths in the
visible region of the spectrum used sources of excitation excite at
laser line 488 nm. Therefore, that exemplary excitation wavelength
will be used for purposes of the description of the invention.
Those in the art will recognize that other energy transfer labels
can be made for specific excitation sources without departing from
the scope of this invention.
The energy transfer between donor and acceptor fluorochromes that
are appropriately selected and linked can be very efficient. The
complexes prepared and described herein show energy transfer
ranging from 50 to 99% efficiency. Energy transfer efficiency
depends on several factors such as spectral overlap, spatial
separation between donor and acceptor, relative orientation of
donor and acceptor molecules, quantum yield of the donor and
excited state lifetime of the donor.
Complexes may be constructed using rigid linkers that optimally
orient the transition moments of the donor and acceptor
chromophores. Alternatively, the linker may be flexible. For
optimal energy transfer to occur, the transition moments of the
first and second fluorochromes are oriented relative to each other
in a nonperpendicular direction. Translation moments positioned
generally parallel or in tandem relative to each other provide
efficient transfer. In practice, the fluorochromes are not in a
static position. The nonrigid linker covalently binding the
fluorochromes flexes, particularly when the complexes are in
solution. The transition moments of the fluorochromes will change
as the linker flexes, but, provided the donor and acceptor
transition moments are nonperpendicular during the excited state
lifetime of the donor, energy transfer will occur.
Shorter linkers would enhance transfer, since efficiency varies as
the inverse 6th power of separation of the centers of the
chromophores according to Forster's Equation:
where ET is energy transfer; K.sup.2 is the relative orientation of
donor and acceptor transition moments; .phi..sub.D is the quantum
yield of the donor molecule; R is the distance between the centers
of the donor and acceptor fluorochromes; J is the overlap between
the emission spectrum of the donor and the absorption spectrum of
the acceptor fluorochromes; and .tau..sub.D is the excitated state
life time of the donor molecule. See, Forster, T., "Intermolecular
Energy Transfer and Fluorescence," Ann. Physik., vol. 2, p. 55
(1948). The distance R between the centers of the two
fluorochromes, e.g., in a complex having two cyanine dyes, the
middle of the methine bridge of one cyanine to the middle of the
methine bridge of the second cyanine, along the length of the
compounds may be from 10 to about 80 Angstroms. The length of the
linker connecting the fluorochromes, as used herein, is different
from the distance R. The linker should permit resonance energy
transfer between the fluorochromes. The fluorochromes should not
interact chemically or form secondary bonds with each other. A
preferred length for the linker is less than 70 Angstroms (7 nm),
and more preferably, less than 20 Angstroms (2 nm). In terms of
bond length, the linker may be from 2 to 20 bond lengths. For
example, if the linker includes an alkyl chain, --(CH.sub.2).sub.n
--, the carbon number n may be from 1 to about 15. As n exceeds 15,
the efficiency of the energy transfer decreases. The linker may
include part of the constituents extending from the cyanine dye. In
other words, the linker is attached to the dye chromophore, but is
not apart of it. For example, referring to the linker shown in
Table 1, some extend from the ring N in one cyanine to a functional
group on the benzene ring of the other cyanine. Some extend between
functional groups on the benzene rings of linked dyes. The linker
is placed on one cyanine dye before the dye linker combination is
attached to the second dye. With a relatively short linker and
optimal orientation, there may be efficient resonance energy
transfer even when the spectral overlap becomes small. Therefore,
it is possible to obtain large wavelength shifts even when only two
chromophores are used in the complex.
The fluorescent labeling complexes include groups capable of
forming covalent bonds with corresponding groups on target
compounds. Preferably, reactive groups are on the complex and
functional groups are on the target compound or molecule. However,
those skilled in the art will recognize that the functional groups
may be placed on the complex and the reactive groups may be on the
target.
The reactive groups of the complexes of the invention include
succinimidyl esters, isothiocyanates, dichlorotriazine, isocyanate,
iodoacetamide, maleimide, sulfonyl halide, alkylimidoester,
arylimidoester, carbodiimide, substituted hydrazines,
hydroxylamines, acid halides and phosphoramidite. The reactive
groups will form covalent bonds with one or more of the following
functional groups: amine, hydroxyl sulfhydryl, carboxyl and
carbonyl.
To promote water solubility, water solubilizing constituents may be
attached to the complex or to the linker. They include amide,
sulfonate, sulfate, phosphate, quaternary ammonium, hydroxyl and
phosphonate groups. Sulfonate or sulfuric acid groups attached
directly to the aromatic ring of the cyanine fluorochrome are
preferred.
Examples of some of the dyes that can be used as donor and acceptor
fluorochromes in the fluorescent labeling complexes of the
invention are shown in Table 1 below:
TABLE 1
__________________________________________________________________________
##STR1## CASCADE BLUE ##STR2## FITC ##STR3## CY3NH.sub.2 ##STR4##
CY3(NH.sub.2).sub.2 ##STR5## CY3NH.sub.2 SO.sub.3 ##STR6##
CY3-O(SO.sub.3).sub.2 ##STR7## CY5(SO.sub.3).sub.2 ##STR8##
CY5(COOH) ##STR9## CY7(SO.sub.3).sub.2
__________________________________________________________________________
Additional cyanines for use in the complexes of the invention are
the rigidized monomethine cyanines disclosed in the copending
applications of the Ratnakar Majumdar. Bhalchandra Karandiker and
Alan S. Waggoner entitled "Rigidized Monomethine Cyanines" Ser. No.
08/474,056 filed Jun. 7, 1995, and "Monomethine Cyanine Rigidized
by a Two-Carbon Chain", Ser. No. 08/474,057 filed Jun. 7, 1995 now
U.S. Pat. No. 5,852,191, the disclosures of which are incorporated
herein by reference. The monomethine rigidized dyes have the
general structures ##STR10## The boron rigidized monomethine
cyanine dyes have sharp distinct absorptive and emissive signals,
and are photostable. Certain of the boron-rigidized monomethine
cyanines maximally absorb and emit light at wavelengths between
400-500 nm or less and fluoresce in the blue region of the visible
spectrum.
Experiments have demonstrated that for obtaining exceptionally
large excitation-emission wavelength shifts, it is possible to use
sequential energy transfer steps in the complex maximal emission at
the wavelength of a cyanine dye, the heptamethine cyanine
designated CY7 in Table 1 above (780 nm) with excitation at 488 nm.
The initial donor was fluorescein and the intermediate fluorophore
in this complex was a trimethine cyanine dye designated generally
as CY3. The fluorescein was excited at 488 nm and transferred
nearly 100% of its excited state energy to the trimethine cyanine,
which in turn transferred about 90% of its excited state energy to
the CY7, fluorescing at 782 nm. The same efficiency was observed
when a pentamethine cyanine, CY5 was used in place of CY7, with
fluorescence at 667 nm. The development of such multichromophore
complexes is particularly useful for multicolor detection
systems.
Although several of the complexes show efficient energy transfer,
the overall quantum yield of these labeling complexes can be
further improved. For example, the use of acceptor dyes with
quantum yields higher than that of CY5 (see Table 1) would improve
the overall "brightness" of the complex.
An experiment was done to determine if two cyanine fluorochromes
could be covalently linked for energy transfer. The cyanines used
were CY5 and CY7 without reactive groups. The results demonstrated
that the cyanines could be covalently linked. The procedure is
presented schematically below. The dyes are represented by
boxes.
EXAMPLE 1 ##STR11## 1. 5 mg cyanuric chloride (trichlorotriazine),
2 mg NaHCO.sub.3, and 0.25 mL purified dimethyl formamide (DMF) as
solvent were mixed at 0.degree. C. To this solution was added 5 mg
of amino-cyanine-5 dye represented above by the box containing
number 5, and the mixture was stirred at 0.degree. C. for 10 min.
Stirring was continued overnight at room temperature. Thin layer
chromatography (TLC) revealed one major spot and two minor spots;
the latter spots were determined to be impurities. UV-visible
absorption showed a peak at 664 nm with a shoulder at 605 nm.
2. The reaction mixture was worked up by precipitation with ether.
A dark blue powder was obtained. 0.3 mL DMF was added to dissolve
the powder. Then 2 mg of sodium bicarbonate and 4.7 mg of amino-CY7
dye, represented above by the box containing number 7, were added.
The mixture was stirred at room temperature for 24 hrs. Absorption
peaks showed at 650 nm (with a shoulder at 607 nm) and 761 nm. The
reaction was precipitated by several washes with ether, providing a
dark powder.
Following the initial success of the above experiments, six energy
donor-acceptor complexes were prepared from cyanine fluorochromes
in order to investigate the energy transfer efficiency of such
compounds. The structures of these complexes are shown in Table 2
and their spectral properties are described in Table 3.
TABLE 2
__________________________________________________________________________
##STR12## 1 ##STR13## 2 ##STR14## 3 ##STR15## 4 ##STR16## 5
##STR17##
__________________________________________________________________________
"A" designates the fluorochrome that acts as the energy acceptor
and "D" designates the fluorochrome that acts as the energy
donor.
The energy transfer complexes shown in Table 2 are as follows:
Complex 1, CY3NH.sub.2 SO.sub.3 (Donor)+CY5(SO.sub.3).sub.2
(Acceptor); Complex 2, CY3--O(SO.sub.3).sub.2 (Donor)+CY3NH.sub.2
(Acceptor); Complex 3, CY3NH.sub.2 (Donor)+CY5COOH (Acceptor);
Complex 4, CY3NH.sub.2 (Donor)+CY5(SO.sub.3).sub.2 (Acceptor);
Complex 5, CY3(NH.sub.2).sub.2 (Donor)+CY7(SO.sub.3).sub.2
(Acceptor); Complex 6, 2 CY3NH.sub.2 SO.sub.3
(Donor)+CY5(SO.sub.3).sub.2 (Acceptor)
TABLE 3 ______________________________________ Spectral Properties
Of Cyanine Dyes Used As Precursors For The Fluorescent Energy
Transfer Labeling Complexes Of The Invention Absorption Emission
Quantum maximum maximum yield Dye Solvent (nm) (nm) (.PHI.)
______________________________________ Amine containing Cyanine
Dyes CY3NH.sub.2 Methanol 552 569 0.05 PBS 548 563 0.05 CY3
(NH.sub.2).sub.2 Methanol 552 569 0.05 PBS 548 563 0.05 CY3NH.sub.2
SO.sub.3 Methanol 556 573 0.08 PBS 548 653 0.09 Carboxyalkyl
containing Cyanine Dyes CY5COOH Methanol 658 685 0.22 PBS 648 667
0.13 CY5 (SO.sub.3).sub.2 Methanol 658 677 0.4 PBS 650 667 0.27
CY3O (SO.sub.3).sub.2 Methanol 492 506 0.20 PBS 486 500 0.09 CY7
(SO.sub.3).sub.2 Methanol 758 789 ND.sup.a PBS 750 777 ND.sup.a
______________________________________ .sup.a N.D. means not
determined. PBS means phosphatebuffered saline.
The efficiency of energy transfer was estimated by calculating the
amount of quenching of donor fluorescence that occurs (DQE) when
the acceptor is attached. It is possible that some quenching could
occur by pathways other than resonance energy transfer when the
acceptor is bound. However, the cyanine donor preferred for the
fluorescent labeling complexes of the present invention are
relatively insensitive to their molecular environment. Furthermore,
addition of large substituents to trimethine cyanines usually
increase, rather than decreases, their fluorescence. Therefore, DQE
may be equal to the efficiency of energy transfer. The estimated
energy transfer efficiencies based on DQE measurements ranged from
50% to 99% and the wavelength shifts between the donor absorption
maxima and the terminal acceptor emission maxima (DI) varied
between 83 nm and 294 nm.
Two of the complexes, 1 and 6, are capable of absorbing light at
the argon laser wavelength, 488 nm. Complex 1 contains a single
donor and single acceptor, and complex 6 contains 2 donors per
acceptor. Complex 1 has 3 carboxyl groups and complex 6 has 4
carboxyl groups. These are converted to succinimidyl active esters
upon activation. FIG. 3 shows the absorption spectra of complex 1
and complex 6 in methanol. The spectra of the complexes are almost
superimposable on absorption spectra obtaining by mixing 1:1 and
2:1 parts of the individual fluorochromes, CY3 and CY5,
respectively, as shown in FIGS. 2(a) and 2(b).
Complex 1 was selected for further studies. As shown in FIGS. 4a
and b, the absorbance (solid line) of complex 1 varies slightly in
phosphate buffer saline (FIG. 4b) and methanol (FIG 4a) but
fluorescence remains unchanged. The emission of the donor component
at 572 nm is very weak compared with the emission of the acceptor
at 675 nm, as would be expected when energy transfer is
efficient.
FIG. 6 demonstrates that sheep antibodies can be readily labeled
with the activated complex 1. Conjugates made of complex 1
conjugated to sheep IgG at various dye:protein rations were tested.
The lowest dye:protein ratio is represented by the line having its
first peak (at about 270 nm) at 0.8 and the highest dye:protein
ratio is represented by the line having its first peak (at about
270 nm) at a little less than 0.4. No dimer formation involving
either the donor or the acceptor fluorochromes was observed with
increasing dye:protein ratios. Each complex 1 contains up to 3
reactive groups. More reactive groups may be used provided no
cross-liking occurs. It is important to use labeling conditions
that avoid protein cross-linking which quench the fluorescence.
Cross-linking by doubly activated cyanines has been observed
previously by Southwick, P. L. et al., "Cyanine Dye Labeling
Reagents: Carboxymethylindocyanine succinimidyl esters," Cytometry,
vol. 11, pp. 418-430 (1990) and can be minimized by limiting the
concentration of protein to be labeled to approximately 1
mg/mL.
Upon binding to antibodies, the quantum yield of the complex was
enhanced three fold as shown in Table 4.
TABLE 4
__________________________________________________________________________
Spectral Properties Of Energy Transfer Complexes Absmax Excitation
Quantum Energy Wavelength nm Wavelength Emmax Yield transferred
Shift Dye (ex 10.sup.4) (nm) (nm) (.PHI.) (%) (nm)
__________________________________________________________________________
Complex 1.sup.a 556 (9.5), 652 (14.3) 488 675 0.32 91 119 514 676
0.37 92 120 600 673 0.49 -- -- Complex 1.sup.b 536 (16); 658 (16)
488 675 0.03 89 139 514 673 0.04 89 137 600 668 0.21 -- -- Complex
558, 658 488 674 0.11 95 116 1.sup.PBS,c 514 673 0.13 95 116 600
676 0.14 -- -- Complex 1.sup.d 562, 658- 488 674 0.19 514 674 0.32
600 674 0.39 Complex 2.sup.a 490 (13), 554 (9.5) 466 571 0.15 89 81
Complex 3.sup.a 545 (9.5), 658 (14.3) 514 679 0.08 83 133 Complex
4.sup.a 550 (9.4), 656 (14.2) 514 674 0.2 96 124 Complex 5.sup.a
445 (9.5), 754 (14.4) 520 782 N.D. 99 226 Complex 6.sup.a 556
(9.5), 652 (14.4) 488 674 0.23 49 118 514 674 0.24 50 118 600 674
0.34 -- -- Complex 6.sup.b 548 (20.0), 652 (15.0) 488 566 0.05 43
118 514 564 0.05 38 116 600 668 0.23 -- --
__________________________________________________________________________
.sup.a In methanol, .sup.b In PBS .sup.c Complex 1 on streptavidin,
d/p = 4 .sup.d In glycerine N.D. means not determined
It is believed that this occurs because the radiationless
deactivation pathway of both the CY3 and CY5 components of complex
1 are reduced because of their restricted mobility when bound to
the surface of the protein. Other means of restricting
conformational mobility are known to increase the fluorescence
efficiency of cyanine fluorochromes, as described in Mujumdar, R.
B. et al. "Cyanine dye labeling reagents. Sulfoindocyanine
succininmidyl ester," Bioconjugate Chemistry, vol. 4, pp. 105-111
(1993). In fact, when complex 1 was dissolved in glycerine, the
quantum yield increased by several fold as shown in Table 3.
Activated complex 1 can be used as a fluorescence label for 2 color
flow cytometry experiments with 488 nm excitation. The scatter plot
is shown in FIG. 7. Human T-lymphocytes were used to compare the
complex 1 label with another 2-color reagent, R-Phycoerythrin,
which also excites at 488 nm and emits at 575 nm. Complex 1 labeled
streptavidin (fluorochrome/protein.about.4) was used to detect
biotinylated CD3 antibody, which marks all T-cells. In the same
lymphocyte sample, Phycoerythrin(PE)-labeled anti-CD4 was used to
mark the Helper-Cell subset of the T-Cells. Thus, in the total
lymphocyte population there is a population of cells that contain
neither CD3 nor CD4 (i.e., CD3 and CD4 negative, shown in the lower
left population of the 2-dimensional scatter plot in FIG. 7), a
subset of complex 1--labeled CD3--positive cells that do not have a
Phycoerythrin signal (i.e., CD3 positive and CD4 negative, shown in
the upper left population of FIG. 7), and a third subset consisting
of complex 1 labeled cells that are Phycoerythrin stained (i.e.,
CD3 and CD4 positive, shown in the upper right population of FIG.
7). It is clear that complex 1 gave base-line separation of the
positive and negative cell populations, and that there was no spill
over of complex 1 fluorescence into the Phycoerythrin channel. The
complex 1 fluorochrome gave a three time brighter signal when the
fluorochrome was excited at 514 nm.
The method of synthesizing complex 1 is described in the example
below.
EXAMPLE 2
Purification of Dyes: Purification of the fluorochromes was
performed on a Spectra-Physics model SP8700 analytical HPLC unit
equipped with a C8-RP column. Purification could also be achieved
by conventional or flash column chromatography on commercially
available C18-RP powder. Water-methanol mixtures were used for
elution in all experiments. Dyes were recovered form the fractions
with a rotary evaporator at 60-70.degree. C. without appreciable
loss. The fluorochrome was passed with unknown counterion
composition through a Dowex-50W hydrogen, strongly acidic cation
exchange column that had been previously washed with 0.1N sulfuric
acid and then distilled water for further purification.
Spectroscopic Measurements and Analytical Determinations:
Ultraviolet-visible spectra were measured with a Hewlett-Packard HP
8452 diode array spectrophotometer. The proton NMR spectra were
obtained with an IBM 300 FT-NMR spectrometer using D.sub.2 O or
DMSO d.sub.6 as solvent. Fluorescence measurements were performed
by using a SPEX Fluorolog 2 system. Quantum yields were determined
by well known techniques as previously described in Mujumdar, R. B.
et al., "Cyanine Dye Labeling Reagents Containing Isothiocyanate
Groups," Cytometry, vol. 10, pp. 11-19 (1989). NMR signals are
described in .delta. by use of s for singlet, d for doublet, t for
triplet and q for quartet, and m for multiplet.
Cell preparation and flow cytometry: Mononuclear leukocytes were
obtained by Histopaque density 1.077 separation of peripheral blood
from healthy volunteers. The lymphocyte population was selected by
flow cytometry based on forward and side scatter characteristics.
Subpopulations were identified using specific monoclonal antibodies
(CD4, staining T-helper cells and CD3, pan T-cell population).
Optimal concentration of complex 1 tagged antibody was determined
by analyzing the results of a dilution series. Direct
immunofluorescence was accomplished by incubating the recommended
amount of labeled antibody with 1-2.times.10.sup.6 cells for 45
minutes at 4.degree. C. Samples were then washed twice in Hank's
balanced salt solution (HBSS) containing 2% fetal bovine serum and
0.1% sodium azide. After the final wash, the cells were resuspended
in 1 mL of HBSS containing 1% paraformaldehyde and analyzed within
one week. Flow cytometry measurements were made with a Becton
Dickinson FACS 440 dual laser flow cytometer equipped with a
Consort 40 data analysis system. The argon ion laser provided 400
mW of excitation at 488 nm. Fluorescence
signals from complex 1 and R-Physoerythrin were collected using
670/13.5 nm and 575/26 nm band pass filters, respectively.
Calculation of donor quenching efficiency (DOE): Absorption and
fluorescence spectra of the donor (alone) and the fluorescent
labeling complex were obtained in order to determine the relative
concentrations of each in fluorescence experiments. Donor
excitation was used to obtain emission spectra of both compounds.
DQE was then calculated using
where F is the fluorescence intensity of the donor alone, F.sup.ET
is the intensity of the donor of the complex, A is the absorbance
at the wavelength of excitation (488 nm) of the donor alone and
A.sup.ET is the absorbance at the wavelength of excitation (488 nm)
of the fluorescent labeling complex.
Syntheses of fluorochromes: Amino-cyanines (CY3NH.sub.2,
CY3(NH.sub.2).sub.2 & CY3NH.sub.2 SO.sub.3) and carboxyalkyl
cyanines (CY5COOH, CY3O(SO.sub.3).sub.2, CY5(SO.sub.3).sub.2)
required as precursors for energy transfer fluorochromes were
synthesized by the methods previously described in Ernst, L. A. et
al., "Cyanine Dye Labeling Reagents For Sulfhydryl Groups",
Cytometry, vol. 10, pp. 3-10 (1989), Hammer F. M., THE CYANINE DYES
AND RELATED COMPOUNDS, (Wiley pub. New York, 1964), Mujumdar, R. B.
et al., "Cyanine Dye Labeling Reagents Containing Isothiocyanate
Groups," Cytometry, vol. 10, pp. 11-19 (1989), Mujumdar, R. B. et
al. "Cyanine dye labeling reagents. Sulfoindocyanine succininmidyl
ester," Bioconjugate Chemistry, vol. 4, pp. 105-111 (1993), and
Southwick, P. L. et al., "Cyanine Dye Labeling Reagents:
Carboxymethylindocyanine succinimidyl ester," Cytometry, vol. 11,
pp. 418-430 (1990). The synthesis and properties of one
amino-cyanine fluorochrome, CY3NH.sub.2 SO.sub.3, and its
conjugation with the succinimidyl ester of CY5(SO.sub.3).sub.2 to
form complex 1 is described below. The spectral properties for all
the fluorochromes are shown in Table 3 and 4 above. The
unsymmetrical trimethinecarbocyanine, CY3-NH.sub.2 SO.sub.3, was
synthesized in four steps. Refer to Table 5 below for the structure
(I)-(VI).
TABLE 5 ______________________________________ ##STR18## R.sub.1
R.sub.2 X ______________________________________ I H H Br.sup.- II
CH.sub.2 Phth H Br.sup.- III CH.sub.2 Phth (CH.sub.2).sub.5 COOH
Br.sup.- IV SO.sub.3.spsb.- (CH.sub.2).sub.5 COOH --
______________________________________ ##STR19## V, R.sub.1 =
SO.sub.3.sup.-, R.sub.2 = CH.sub.2 Phth VI, R.sub.1 =
SO.sub.3.sup.-, R.sub.2 = CH.sub.2 ((CY3NH.sub.2 SO.sub.3)
______________________________________ ##STR20## - 1. Synthesis of
5-Phthalimidomethyl-1-(.epsilon.-carboxypentynyl)-2,3,3-trimethylindole,
(III). 5-Phthalimidomethyl-2,3,3-trimethylindolenine (II) was
synthesized according to the procedure of Gale and Wilshire, "The
amidomethylation and bromination of Fisher's base. The preparation
of some new polymethine dyes," Aust. J. Chem., vol. 30, pp. 689-694
(1977). Powdered N-hydroxymethylphthalimide (70 g, 0.4 mol) was
added in small portion over a period of 45 min. to a stirred
solution of 2,3,3-trimethyl-(3H)-indolenine (I), (70 g, 0.44 mol)
in concentrated sulfuric acid (360 mL) at room temperature. The
solution was stirred for 70 h at room temperature before being
poured onto ice-water. Basification of the solution with conc.
ammonium hydroxide gave a yellow powder which was filtered and
dried. (111 g, yield 80%, m.p. 180-182.degree. C.). .sup.1 H NMR
(DMSO d.sub.6), .delta., 7.8-7.95 (m, 4H, phthalimido), 7.4 (s, 1H,
4-H), 7.38 (d, 1H, J=9.0 Hz, 6-H), 7.2 (d, 1H, J=9 Hz, 7-H), 4.7
(s, 2H, --CH.sub.2), 2.2 (s, 3H, CH3), 1.2 (s, 6H
--(CH.sub.3).sub.2).
This dry powder (10 g, 0.3 mol) and 6-bromohexanoic acid (9.1 g,
0.05 mol) were mixed in 1.2-dichlorobenzene (25 mL) and heated at
125.degree. C.) for 12 h under nitrogen. The mixture was cooled,
1,2-dichlorobenzene was decanted and the solid mass was triturated
with isopropanol until free powder was obtained. (11 g, yield 80%,
m.p. 124-126.degree. C.). .sup.1 H NMR (DMSO d6), .delta., 7.8-7.95
(m, 4H, phthalimido), 7.4 (s, 1H, 4-H), 7.38 (d, 1H, J=9.0 Hz,
6-H), 7.2 (d, 1H, J=9 Hz, 7-H), 4.7 (s, 2H, --CH.sub.2), 4.5 (t,
2H, J=7.5 Hz, .alpha.-CH.sub.2), 2.3 (t, 2H, J=7 Hz,
.epsilon.-CH.sub.2), 1.99 (m, 2H, .beta.-CH.sub.2), 2.3-1.7 (m, 4H,
.gamma.-CH.sub.2 and .delta.-CH.sub.2 merged with s of
6H--(CH.sub.3).sub.2).
2. Synthesis of
1-(.delta.-Carboxypentynyl)-2,3,3-trimethylindoleninium-5-sulfonate
(IV). Compound (IV) was synthesized according to the procedure
described previously by Mujumdar, R. B. et al., Bioconjugate
Chemistry, (1993), supra. The potassium salt of
2,3,3-trimethylindoleninium-t-sulfonate (11 g, 0.04 mol) and
6-bromohexanoic acid (9.8 g, 0.05 mol) were mixed in 1,2
dichlorobenzene (100 mL) and heated at 110.degree. C. for 12 h
under nitrogen. The mixture was cooled. 1,2-dichlorobenzene was
decanted and the solid mass was triturated with isopropanol until
free powder was obtained, (11 g, yield 80%) .lambda.max (water) 275
nm: .sup.1 H NMR (D.sub.2 O) .delta. 8.13 (s, 1H, 4-H, 8.03 (dd,
1H, J=9.0, 1.1 Hz, 6-H), 7.2 (d, 1H, J=9.0 Hz, 7-H), 4.51 (t, 2H,
J=7.5 Hz, .alpha.-CH.sub.2), 2.25 (t, 2H, J=7.5 Hz,
.gamma.-CH2.sub.2), 1.99 (m, 2H, .beta.-CH2--), 1.35-1.66 (m, 4H,
.delta.-CH.sub.2, .gamma.-CH.sub.2), 1.61 (s, 6H,
--(CH.sub.3).sub.2). R.sub.f =0.55 (C-18, water-methanol, 25%).
3. Synthesis of Intermediate: A solution of
1-(.gamma.-carboxypentynyl)-2,3,3-trimethylindoleninium-5-sulfonate
(IV) (10 g, 0.03 mol) and N,N' diphenylformamidine (7.2 g, 0.04
mol) in acetic acid (20 mL) was heated to reflux for 1 h. Acetic
acid was removed on a rotary evaporator and the product washed with
ethyl acetate (3.times.50 mL) whereupon a dark brown solid was
obtained. .lambda.max (water) 415 nm, R.sub.f =0.32 (C18, 25%
methanol in water). The crude product thus obtained was used for
the next reaction without further purification. The solid (3.8 g)
was dissolved in a mixture of acetic anhydride (10 mL) and,
pyridine (5 mL).
5-Phthalimidomethyl-1-(.epsilon.-carboxypentynyl)-2,3,3-trimethylindole,
(III) (2.6 g, 6 mmol) was added and the reaction mixture was heated
at 110.degree. C. for 1 h. The solution was cooled and diluted with
several volumes of diethyl ether (500 mL). Product separated in the
form of red powder from which supernatant fluid was removed by
decantation. It was dissolved in a minimum volume of methanol and
reprecipitated with 2-propanol. The product was collected on a
filter paper and dried to yield 5.3 g of compound (V). It was
purified by flash column chromatography on reverse-phase C18 using
a water-methanol mixture as eluent, (1.6 g, yield 30%). .lambda.max
(water) 554 nm, .epsilon. 1.3.times.10.sup.5 L/mol-cm. .sup.1 H NMR
(CD.sub.3 OD) .delta. 8.5 (t, 1H, J=14 Hz, .beta.-proton of the
bride); 7.8-8.0 (m, 6H, 4 protons of phthalimido group & 4-H
& 6-H of sulfoindole ring), 7.55 (s, 2H, 4'-H); 7.6 (d, 1H,
J=12 Hz, 6'-H); 7.3 (two d. 2H, 7-H & 7'-H); 6.1-6.3 (t, 2H,
.alpha..alpha.' protons of the bridge); 4.1 (m, 4H, .alpha. &
.alpha.'--CH.sub.2 --); 2.9 (t, 2H, J=7 Hz, --CH.sub.2 COOH);
1.4-2.0 (m, 21H, three --CH.sub.2, one --CH.sub.3 and two
--(CH.sub.3).sub.2), methyl protons of the methylphthalimido group
are merged in a water signal at 4.8.
4. Hydrolysis of (V) to Give (VI). (1 g. 1.1 mmol) was dissolved in
concentrated hydrochloric acid (5 mL) and heated under reflux for
12 h. After cooling, the crystalline phthalic acid was filtered
off. The filtrate was concentrated with a rotary evaporator and
then slowly neutralized with concentrated ammonium hydroxide while
the temperature was kept below 30.degree. C. Pure fluorochrome
CY3NH.sub.2 SO.sub.3 (VI) was obtained by reverse phase (C18)
column chromatography using a water-methanol mixture as eluent.
.lambda.max (methanol) 552 nm, .sup.1 H NMR (DMSO, d.sub.6) .delta.
8.45 (t, J=7.2 Hz, 1H, 9-H); 7.3-7.9 (m, 6H, aromatic protons);
6.55 (dd, 2H, 8 & 8'-H); 4.5 (m, 4H, N--CH.sub.2); 4.1 (s, 2H,
CH.sub.2 NH.sub.2); 2.15 (t, 2H, CH.sub.2 COOH); 1.25-1.8 (broad m,
24H, 2-(CH.sub.2)2 & 6-C--(CH.sub.3).sub.2). R.sub.f 0.415 (RP
C18 60% methanol in water).
5. Synthesis of complex 1. Dry powder of CY5(SO.sub.3).sub.2
succinimidylester (425 mg, 0.26 mmol) was added in small portions
to a well stirred solution of CY3NH.sub.2 SO.sub.3 (200 mg, 0.26 m
moles) in 10 mL of carbonate-bicarbonate buffer (0.1M, pH 9.4).
Stirring was continued for additional 30 minutes after which the
reaction mixture was purified by flash column chromatography on C18
reverse phase powder using water:methanol (6.3:3.7) as solvent as
eluent. 5 mL fractions were collected and monitored by TLC.
Fractions containing CY5(SO.sub.3).sub.2 acid and CY3NH.sub.2
SO.sub.3 were discarded. Violet colored fractions were checked by
ultraviolet light in methanol and the fractions containing complex
1 fluorochrome (FIG. 3) were pooled. Evaporation of the solvent
yielded 150 mg of complex 1 as violet powder (37%) R.sub.f =0.45
(RP 37% methanol in water) fluorochrome 1:1 yield 37%. .sup.1 H NMR
spectrum recorded in D.sub.2 O showed broad signals and were
difficult to assign. The fluorochrome was purified on a strongly
acidic ion-exchange column (Dowex 50) to remove cationic counter
ions. High resolution FAB mass spectrometry showed (M+H).sup.+ ion
at 1391.83 C.sub.73 H.sub.91 N.sub.5 O.sub.16 S.sub.3 +H requires
1391.73).
6. Succinimidyl Ester of Energy Transfer Cyanine Dye Complex 1 (60
mg, 0.04 mmol) was dissolved in a mixture of dry DMF (1 mL), and
dry pyridine (0.05 mL). Disuccinimidyl carbonate (DSC) (46 mg, 0.18
mol 1.5 equiv/carboxyl group was added and the mixture was stirred
at 55-60.degree. C. for 90 min. under nitrogen. After diluting the
mixture with dry diethyl ether (20 mL), the supernatant was
decanted. The product was washed repeatedly with ether, filtered
and dried under vacuum. The formation of the active succinimidyl
ester was confirmed by its reaction with benzylamine in DMF or its
reaction with taurine in a pH 9.4 bicarbonate buffer. Reversed
phase C18 TLC spotted with the conjugate, the succinimidyl ester
and hydrolyzed carboxylate product for comparison was developed
with water-methanol (1:1) mixture. R.sub.f =0.78 (Acid), 0.3
(Benzylamine adduct).
7. Reaction of Succinimidyl Ester with Antibody and Streptavidin. A
stock solution of the complex 1 fluorochrome succinimidyl active
ester was made in dry DMF (1 mg/100 mL). In one sample, one
milligram Sheep .gamma.-globulin was dissolved in 0.25 mL
carbonate/bicarbonate buffer (approximately 6.45 mmol/0.25 mL). In
another, streptavidin was dissolved in 0.25 mL of the
carbonate/bicarbonate buffer. Appropriate volumes of the
fluorochrome stock were added to 0.25 mL portions of each protein
solution to produce desired starting fluorochrome to antibody
ratios, and each reaction mixture was stirred at room temperature
for 30 minutes. The protein conjugate was separated from unreacted
fluorochrome in each sample by gel filtration chromatography over
sephadex G-50 (0.7.times.20 cm column), using PBS, pH 7.4,
containing 0.1% azide. Dye conjugated proteins eluted as colored
bands well separated from the unreacted fluorochrome. The
normalized spectrum of the complex 1-streptavidin conjugage in PBS
is shown in FIG. 5. The absorbance spectrum of complex 1--Sheep IgG
in PBS is shown in FIG. 6. FIG. 7 shows the flow cytometry analysis
of complex 1-streptavidin used to detect CD3 antibody.
EXAMPLE 3
Several other complexes were synthesized.
FIGS. 8 and 9 shown the absorbance and emission spectra,
respectively, for the complex fluorescein-CY3NH.sub.2 SO.sub.3 in
methanol having the structure. ##STR21## Excitation was at 488 nm
with fluorescence emission at 574 nm. The quantum yield was 0.041,
the Stokes shift was 74 and the % efficiency of the energy transfer
was 98.3%. The absorbance max. for each of the fluorochromes in the
complex is 500 and 558. FIGS. 10 and 11 shown the absorbance and
emission spectra, respectively, for the complex
fluorescein-CY3(NH.sub.2).sub.2 --CY5(SO.sub.3).sub.2 in methanol.
The absorbance max. for each of the fluorochromes is 500, 560 and
650. Excitation was at 488 nm and emission at 672 nm. The quantum
yield was 0.1566, Stokes shift was 172 and the % efficiency of the
energy transfer was 99%. FIGS. 12 and 13 shown the absorbance and
emission spectra, respectively, for the complex
fluorescein-CY3(NH.sub.2).sub.2 --CY7(SO.sub.3).sub.2. The
absorbance max. for each fluorochrome is 500, 560 and 754.
Excitation was as 488 nm and emission at 782 nm. The Stokes shift
was 282 and the % efficiency was 99%. These series of spectra
demonstrate the efficient energy transfer with long Stokes shifts.
Each emission spectrum shows substantially all of the emission
coming from the final acceptor fluorochrome in each series with
only minimal emission from either the donor fluorescein in FIG. 9
or the intermediate cyanine in FIGS. 11 and 13.
Multiparameter analyses can be done of multiple samples to detect
the presence of target biological compounds. Each sample is labeled
by well known labeling methods with a different complex. For
example, one sample suspected of containing a target biological
compound is incubated with a single fluorochrome, such as
fluorescein, cascade blue, a BODIPY dye or one of the monomethine
rigidized dyes or CY3O(SO.sub.3).sub.2 or CY3(SO.sub.3).sub.2, all
emitting in the 500-575 nm wavelength range (green to orange). A
second sample suspected of containing the target biological
compound (the same compound or a different compound as that in
sample 1), is incubated with a complex of the invention, for
example fluorescein-CY3NH.sub.2 which will absorb light at 488 nm
and emits fluorescence at 574 nm (orange). Additional samples
suspected of containing another target compound are incubated with
other labeling complexes of the invention, such as
fluorescein-CY3-CY5 and fluorescein-CY3-CY7 both of which absorb
light at 488 nm, but emit fluorescence at 672 nm and 782 nm,
respectively (red to deep red). After a suitable period to permit
the fluorescent labels to bind with the target compounds, unbound
label is washed and the labeled samples are mixed. Detection is
possible with a single wavelength excitation source, i.e., at laser
line 488 nm. Each differentially labeled sample will fluoresce a
different color at the emission wavelength of its particular label.
Those skilled in the art will recognize that the fluorescent
labeling complexes of the present inventor can be used for a
variety of immunofluorescent techniques, including direct and
indirect immunoassays, or, competitive immunoassays and other known
fluorescence detection methods. The conditions of each incubation,
e.g., pH, temperature and time are known in the art, but generally
room temperature is preferred. If reacting with a amine, pH 9.4 is
preferred. The pH is adjusted depending on the optimum reaction
conditions for the particular reactive groups according to known
techniques.
The fluorescent labeling complexes may be used to form reagents by
covalently binding the complexes to a carrier material, such as
polymer particles, cells, glass beads, antibodies, proteins,
enzymes and nucleotides or nucleic acids (DNA and RNA) and analogs
thereof which have been derivatized to include at least one first
reactive group capable of forming a covalent bond with the
functional group on the labeling complex (or a functional group
capable of forming a covalent bond with a reactive group on the
complex, as described above) and at least one second reactive group
(or functional group, as the case may be) having specificity for,
and being capable of forming a covalent bond with, a target
biological
compound, such as antibodies, cells, drugs, antigens, bacteria,
viruses and other microorganisms. When the carrier has functional
groups, it may be antibody or DNA suited for attachment to antigen
or a complementary DNA sequence, respectively. When the carrier
material has reactive groups on it, the carrier may be a polymer
particle or an antigen suitable for attachment to DNA or an
antibody, for example. Techniques for covalently binding
fluorochromes to carrier molecules such as those mentioned are well
known in the art and readily available in the literature. The
carrier material can further include nucleotide derivatized to
contain one of an amino, sulfhydryl, carboxyl, carbonyl or hydroxyl
groups, and oxy or deoxy polynucleic acids derivatized to contain
one of an amino, sulfhydryl, carboxyl, carbonyl or hydroxyl groups.
The functional groups on the carrier material which are
complementary to, i.e., form covalent bonds with, the reactive
groups of the labeling complexes of the invention include amino,
sulfhydryl, carboxyl, hydroxyl and carbonyl groups.
A comparison of the energy transfer complex of the present
invention to the conventional R-Phycoerythrin dyes is shown in
Table 6 below.
TABLE 6 ______________________________________ COMPLEX 2 vs
R-PHYCOERYTHRIN R-Phycoerythrin Complex 2
______________________________________ Excitation wavelength 488
488 Emission wavelength 580 578 488-laserline PE fluorescence
signals were Flow-Cytometer was greatly stable reduced at pH 8.5
throughout pH & extinguished range at pH 9.5 MW 240000 1667
Staining do not penetrate labeled Ab readily into penetrates
intracellular into tissues to reach intracellular target antigen
tissues to reach target antigen Binding Rate rate of binding rapid
binding to antigen is slow
______________________________________
The energy transfer complexes of the present invention provide a
valuable set of fluorescent labels which are particularly useful
for multiparameter analysis and importantly, are sufficiently low
in molecular weight to permit materials labeled with the
fluorescent complexes to penetrate all structures. As such, the
complexes are well suited for use as DNA probes. The complexes of
the invention and the reagents that can be made from them offer a
wide variety of fluorescent labels with large Stokes shifts. Those
in the art will recognize that the complexes of the invention can
be used in a variety of fluorescence applications over a wide range
of the visible spectrum.
* * * * *